1

Problem Formulation for the Reassessment of Ecological Risks from the Use of Atrazine

Errata Sheet for Appendix C of the White Paper - Open Literature Review of Amphibian Data

While preparing the SAP presentation on evaluation of amphibian toxicity data, discrepancies were observed for some of the endpoint 

classifications as well as some clarifications needed for others. The following table represents the classification changes made to 

endpoints in a study. In all but three cases, the endpoints reported no effects at the highest concentration tested. In all but four cases, 

the reason for the classification change was due to lack of summary data; in the other four cases, endpoints were downgraded as there 

was uncertainty in whether statistical results were analyzed using pseudoreplication which may have changed the outcome of the 

results or in the number of replicates used in the study. Also, all the classification changes were from Qualitative lower level of 

confidence to Invalid or the endpoint was already classified as Invalid. As such, these changes do not change the overall 

understanding of the effects for these endpoints when considering the entire available dataset. 

Table 1. Clarifications of Endpoints 

Citation Endpoint Endpoint Result Reason for Change Classification Change

Changes in Endpoint Classification

Allran and Karasov 

2000

Growth; 

Bio/Mol 

No effect on mass or hematocrit 

@ 192 ppb

Summary data not reported 

(SVL results reported)

Qualitative lower to Invalid

Allran and Karasov 

2001

Growth; 

Behavior; 

Bio/Mol

No effect on mass, swimming, 

hemoglobin @ 20000ppb

Summary data not reported Qualitative lower to Invalid

Clements et al. 1997 Bio/Mol ↑ DNA damage (erythrocyctes) @ 

4800 ppb

Lack of test design (# 

reps)/possible 

pseudoreplication

Qualitative lower to Invalid

Coady et al. 2005 Growth;

Metamorphosis

No effect on mass, SVL or age at 

metamorphosis @ 25 ppb

Summary data not reported Stayed Invalid

Forson and Storfer 

2006b

Growth;

Metamorphosis

No effect on time to 

metamorphosis, mass or SVL @ 

160 ppb

Summary data not reported Qualitative lower to Invalid

Gibble et al. 2011 Bio/Mol No effect on peptide activity @ 

986 ppb

Limited data on test design (# 

reps and animals

Qualitative lower to Invalid

Haridon et al. 1993 Bio/Mol No effect on micronucleated 

RBCs (genotox) at 300 ppb

Uncertain if only one replicate 

used (pseudoreplication)

Qualitative lower to Invalid

Kerby and Stofer Imm/Inf No effects on infection rate @ Summary data not reported Qualitative lower to Invalid
2

Citation Endpoint Endpoint Result Reason for Change Classification Change

2009 200 ppb

Orton et al. 2006 Growth;

Metamorphosis

No effect on SVL or time for 

forelimb emergence @ 15 ppb

Summary data not reported Stayed Invalid

Rohr et al. 2003 Growth No effect on mass @ 400 ppb Summary data not reported 

(SVL results reported)

Qualitative lower to Invalid

Tavera-Mendoza et 

al. 2002a

Sexual 

Development

Effects on male gonadal 

development @ 18 ppb

Uncertain if statistical 

analysis used 

pseudoreplication

Qualitative lower to Invalid

Tavera-Mendoza et 

al. 2002b

Sexual 

Development

Effects on female gonadal 

development @ 18 ppb

Uncertain if statistical 

analysis used 

pseudoreplication

Qualitative lower to Invalid

Williams and 

Semlitsch 2010

Growth No effect on mass @ 1.25 ppb Summary data not reported Qualitative lower to Invalid

Other Endpoint Clarifications

Allran and Karasov 

2001

Behavioral Feeding LOAEC = 20000 ppb; 

NOAEC = 12000 ppb 

Need to add endpoint to study 

summary in Table in Appendix 

C

Qualitative lower

Coady et al. 2005 Metamorphosis Age at metamorphosis NOAEC 

= 25 ppb; LOAEC > 25 ppb

Need to add endpoint to study 

summary in Table in Appendix 

C

Invalid (see above)

Forson and Storfer 

2006a

Imm/Inf Infection rate NOAEC = 184 

ppb; LOAEC > 184 ppb

Need to add endpoint to study 

summary in Table in Appendix 

C

Qualitative lower

Hecker et al. 2005 a Bio/Mol Aromatase, estradiol, CYP19 

NOAEC = 258; LOAEC > 258

Need to add endpoint to study 

summary in Table in Appendix 

C

Qualitative lower

Morgan et al. 1996 Survival Need to remove results reported 

as a.i.

Results already adjusted for 

percent active ingredient

Qualitative medium

Villeneuve et al.

2003

Bio/Mol No effect on gonad and brain 

aromatase @ 25 ppb

Typographical error in 

classification

Qualitative higher to lower
3

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

Allran & Karasov 

2000 / E56572

Rana pipiens / 

Nasco(unknow

n if Lab/field / 

TP (G25) to M 

(138 d total)

T / 

99

NA / 

(Y/NA); 

NS

NS 20 TP/7L;

48 hr

Metam

Gro

Bio/Mol

Surv

192 (M)

192

192

192

>192

>192

>192

>192

% & time to metamorphosis

Mass; SVL

Hematocrit

% mortality

Metamorphosis and growth endpoints based on 

subset (50%) of organisms and uncertainty in 

ability to get a true randomization based on 

information presented; summary data for each 

treatment group for mass, mortality and hematocrit 

not reported (only reported mean & SE for all 

replicates combined); ammonia levels not reported

Qualitative-Lower level of confidence for growth 

and metamorphosis endpoints based on sub 

sampling as cannot discount potential confounding 

factors and also for mortality and hematocrit as no 

treatment summary information present; Invalid 

for mass and hematocrit as summary data not 

presented

Allran & Karasov 

2001 / E59251

R. pipiens & R. 

sylvatica &

Bufo 

americanus / 

Field / A (R. 

pipiens)for 14 d 

and E for 96 hr 

ph

T / 

99

NA / 

(Y/NA); 

NS

Glass (E) / 

NS (A)

50 or 30 

E/0.075L 

(E); 24 hr

Meta

Gro

Behav (E)

Behav (A)

Behav (A)

Gro (A)

Bio/Mol 

(A)

Surv

2590

20000

20000

2590

12000

20000

20000

20000

4320

>20000

>20000

4320

20000

>20000

>20000

>20000

Deformities (tail, facial edema)(E)

Mass (E)

Swimming (E)

Buccal ventilation

↓ Feeding

Mass (A)

Hemoglobin

Hatch and mortality

Atrazine measured at organism collection site; 

water quality parameters not specified-authors 

state conducted according to ASTM; Summary 

data growth,, swimming, hemoglobin not 

presented

Qualitative-Lower level of confidence as low

levels of atrazine measured at organism collection 

site; Invalid for growth, swimming and 

hemoglobin due to lack of summary data

Birge et al. 1980 / 

E6187

R. catesbeiana, 

R. pipiens, R. 

F / 

80

NS / (Y 

unsure if glass 50 -103 E / 0.5L; 2.5 

Surv 4 

dph LC50

-- -- R. catesbeiana = 410

R. pipiens = 7680

LC50s were not based on mortality per se, but on 

abnormalities that would reportedly preclude 

 

1 TP = tadpole; LV = larvae; M= metamorphosis; E= embryo; G = Gosner stage; NF=Nieuwkoop and Faber; SVL=snout-vent-length; A = adults; H = hatch; J = 

juvenile; if reported in study, developmental stage or some other identifier at test initiation is given, if not reported than designated as NS (not specified); 

2 TS = Test substance; T = technical; F = formulation

3 NA = not applicable; NS = not specified; ACTN = acetone; EtOH= ethanol; DMSO=dimethylsulfoxide; MeOH=methanol; IPA = isopropyl alcohol

4 Negative Control (NC) and/or Solvent Control (SC) included in study design; MA in CNT = Measurable atrazine in control treatment group(s)- Yes (Y) or No 

(N) or Not Specified (NS)

5 The values reported represent loading reported as mass (g) or number of larvae/tadpoles in a volume of water (L). If loading was reported as number of 

larvae/tadpoles in a certain test vessel size but test solution volume was not reported, then this is presented # organisms in tank (volume reported if available) as 

actual loading could not be determined; PE = polyethylene

6 Water 

7 Metam = metamorphosis; Gro = growth; Imm/Inf = immune system or infection; Bio/Mol = biochemical/Molecular; Sexdev = sexual development; 

Behav=behavioral; Surv = survival; N = nominal conc.; M = measured conc; NDR = not dose responsive (no significance at higher dose).
4

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

palustris, B. 

americanus / 

NS (purchased) 

/ E for 4 dph

NC or 

SC); NS

h R. palustris = 17960

B. americanus >48000

survival under natural conditions; solvent (if used) 

was not identified; formulation used so uncertainty 

in source of toxicity

Qualitative – Higher level of confidence; however 

LC50 values are combination of both lethality and 

abnormal effects and formulation used

Brodeur et al. 2009 

/ E112588 

R. arernarum / 

LP8/ TP 

(G38/39)-M

T/ 

98

NA / 

(Y/NA9); 

N

glass

10 TP 

/0.04L; 48 

hr

Metam

Surv 

(LC50) 

NDR (N)

NA

100

NA

↓ & ↑Time metamorphic stage and/or 

duration between stages

LC50 (stage 4) = 7150-25370

LC50 (stage 25) = 2320-27160

LC50 (stage 38-39) = 12630-18270

(LC50s range from 4 d to 28 d)

No water quality parameters reported; 

metamorphosis results not dose responsive (not 

sign. at 1000 or 5000 μg/L depending on metam 

endpoint (100 lowest conc tested); hypothesis 

testing for effects on metamorphosis conducted 

using EC50 values 

Qualitative-Lower level of confidence based on 

higher loading with no water quality parameters 

reported

Brodkin et al 2007 / 

E103351

R. pipiens / 

Field (ANA10) / 

A for 8 d

T/ 

NS

NA / (N & 

N

11/NA); 

NS

plastic NA; 24 hr

Imm/inf

Surv

<0.01(N)

10

0.01

>10

↓ White Blood Cells

Study indicated atrazine did not 

cause mortality; not explicitly 

reported in terms of NOEC/LOEC

Adult frogs cultured using Styrofoam rafts; 

uncertainty in how organisms were given what 

treatments; effects are not clearly and 

quantitatively linked to the assessment endpoints 

of growth, reproduction, and survival

Invalid based on lack dilution water only control 

(dilution water but subject to immune treatments 

used)

Choung et al. 

2011a / E153858 

Litoria

raniformis / 

Lab / TP (G26) 

for 10 wks

T/ 

>98

NA / 

(Y/NA); 

NS

glass

10 TP 

/10L; 

2X/wk

Metam

Gro

Surv

25 (N)

25

--

>25

>25

--

Time to metamorphosis

Mass; SVL

Study indicated survival >97.5% for 

study; not explicitly reported in 

terms of NOEC/LOEC 

Limited reporting of potential contaminants in 

food and water; number of egg masses used not 

specified; measured ammonium (not NH3)

Qualitative-Higher level of confidence; however 

there is a lack of reported measurements in food 

water and number of egg masses

Clements et al. 

1997 / E20274

R. catesbeiana / 

CBSC / TP for 

24 hr

F / 

88.2

NA / 

(Y/NA); 

NS

glass NS; NA

Bio/Mol

Surv

<4800 

(4200 ai)

--

4800 

(4200 

ai)

↑ DNA damage in erythrocyctes

NS; reported testing the maximum 

Limited reporting on water quality parameters and 

test design (# of replicates); conducted above 

water solubility

 

8 LP = adults used to collect eggs were obtained from local provider

9 Test concentrations 1-25 mg/L prepared without solvent; 30 mg/L prepared using acetone (0.2%) so solvent control (0.2% acetone) was also included in design

10 ANA= Amphibians of North America (Nashville, TN, USA)

11 A treatment group that was only exposed to dilution water (and not injected with Ringer solution or subjected to thioglycollate stimulation) was not included.
5

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

-- survival concentration and dilutions 

from that conc. Invalid based on lack of test design parameters

Coady et al 2004 / 

46965605

R. clamitans/ 

Field / TP 

(G25)-G42 (273 

d)

T/ 

97.1

EtOH 

(0.005) / 

(Y/Y); Y

NS

30 TP/4L 

then 16L; 

72 hr 

Metam

Gro

Surv

NDR(M)

28

28

11

>28

>28

↑ Time to metamorphosis at 11 but 

not 28

Mass; SVL

% mortality

Poor water quality; high control mortality; lack of 

responsiveness to positive control (estradiol)

Invalid based on control contamination; high 

control mortality

Coady et al 2005 / 

46965602

Xenopus 

laevis/Lab/ TP 

(72 h ph)- M

T/ 

97.1

NA / 

(Y/NA); 

Y

glass

30 TP/4L; 

50% every 

72 hr

Gro

Sexdev

Bio/Mol

Surv

Metam

25

25

0.1

25

25

>25

>25

1

>25

>25

Mass; SVL

Gonad development; laryngeal 

muscle

↓ estradiol males

% mortality

Age at metamorphosis

Higher loading with 50% renewal of solution at 

72 hr may have promoted poor water quality; 

delayed metamorphosis in controls; statistical 

difference between controls (possible confounding 

factor); lack of responsiveness to positive control 

(estradiol); no summary data for growth, 

metamorphosis 

Invalid based on control contamination and for 

growth and metamorphosis for lack of summary 

data

Forson & Storfer 

2006a / E82033

A. 

macrodactylum

/ Field / LV (ca. 

6 wks) for 30 d

T / 

86.5

NA / 

(Y/NA); 

NS

PE

1 LV/ 

0.5L; 

3X/wk

Metam

Gro

Surv

Imm/Inf

18.4 (M)

18.4

184

184

184

184

>184

>184

↑ larval period

↓ mass & SVL

% mortality

Infection Rate

Limited reported water quality;

Qualitative-Lower level of confidence due to lack 

of reported measurements in control with slightly 

higher than recommended loading with limited 

water quality and a renewal rate of 72hr 

periodically

Forson & Storfer 

2006b / E103072

A. tigrinum / 

Lab / LV 

(12wks)-M 

(111 d total)

T / 

NS

NA / 

(Y/NA); 

NS

PE

1 LV 

/0.5L; 

every 3 

days

Metam

Gro

Imm/inf

Surv

160 (N)

160

1.6

(NDR); 

16

--

>160

>160

16;160 

--

Time to metamorphosis

Mass, SVL

↑ infection rate; ↓leukocytes 

Reported 31 mortalities (384 

exposed, 8% mortality); results not 

presented in terms of NOEC/LOEC

Limited reported water quality; infection rate not 

dose responsive (not sign at 160 μg/L) ; potential 

stress from repetitive mass measurements; 

summary data for metamorphosis and growth not 

presented

Qualitative-Lower level of confidence due to 

slightly higher than recommended loading with 

limited water quality and a renewal rate of 72hr; 

Invalid for metamorphosis and growth as 

summary data not presented

Fort et al. 2004 / 

E81751

X. tropicalis &

X. laevis / 

T/ 

NS

DMSO 

(1%) / NS 20 E/0.01L; 

Metam

Surv

-- -- X. laevisLC50 = 24500 or 25600Limited water quality reported; use of not 

recommended solvent and high conc (although 
6

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

Xenopus I

(adults) / E –NF 

46

(Y/NS); 

NS

24 hr (FETAX) EC50 = 4100 or 4400

X. tropicalis

LC50 = 19400 – 31800

EC50 = 2100 - 9300 

allowed under ASTM FETAX assay guideline)

Invalid based on solvent choice and concentration

Freeman & 

Rayburn 2004 / 

E78644

B. americanus 

& X. laevis / 

Field 

(parents)(Bufo) 

& Nasco / TP 

(NF NS) for 3-5

wks

T / 

NS

NS / (C 

used NS if 

NC, SC); 

NS

NS

NS (2 

TP/3L 

tank); 

weekly

Bio/Mol

Surv

800

NS

>800

NS

Nuclei (represent genotox)

Not specified; based on reported ‘N’ 

in tables appears 100% survival

Stats for nuclei conducted on CVs; uncertainty in 

sample sizes; no reported water quality; loading 

unknown (fill volume not reported)

Qualitative-Lower level of confidence due to 

uncertainty in sample sizes and loading unknown 

with no reported water quality and relatively long 

renewal period

Freeman & 

Rayburn 2005 / 

E81459

X. laevis / 

Nasco/Xenopus 

Express / TP 

(varied NF40 to 

54) for 3-5 wks

T / 

98

NA / 

(Y/NA); 

NS12

NS

NS (1 

TP/3L 

tank); 

weekly

Metam

Gro

Bio/Mol

Surv

<100 (N)

800

Varied

--

100

>800

800

--

↓ metamorphic stage

Mass

↑ flow cytometric analysis meant to 

illustrate metemorphosis and 

development); results varied based 

on test 

% Mortality (0-16.7%) for different 

assays, reported as occurring 

randomly in treatments; mortality in 

terms of NOEC/LOEC not reported

Stats for flow cytometric conducted on CVs; 

uncertainty in weight analysis with regards to 

accounting for different stages of organisms; fill 

volume of test vessel (3L) not reported however 

water quality appears to be acceptable

Qualitative- Medium level of confidence for 

molecular endpoint due to analysis using CV and 

other reasons described for metamorphosis; 

Invalid for weight due to potential confounding 

factor; Qualitative –Higher –for metamorphosis 

stage; however, lack of analysis of atrazine in 

control

Freeman et al. 2005 

/ E81460

Bufo 

americanus / 

Field (Adults) / 

TP (G20 or 25) 

for 3 or 4 wks

T/ 

98

NA/ 

(Y/NA); 

NS

NS

NS (2 

TP/3L 

tank); 

weekly

Metam

Gro

Bio/Mol

Surv

NDR (N)

NDR

10000

--

250

800

>10000

--

↑ Stage at test completion

↓ Mass at test termination

Mean total # of nuclei 

Reports no acute toxicity observed; 

mortality in terms of NOEC/LOEC 

not reported

Water quality parameters in study not reported; fill 

volume of test vessel (3L) not reported-unable to 

verify loading; metamorphosis and mass not dose 

responsive (metam-not sign at 800 μg/L in other 

test different age at initiation; mass- not sign at 

800 in other test or 250-10000 in another test)

Qualitative- Lower level of confidence for

Metamorphosis and Bio/Mol- due to loading not 

defined and no water quality parameters with 

weekly renewal; Invalid for weight due to 

potential confounding factor

 

12 Study reports that solutions measured from experimental tanks and atrazine concentrations were within expected range; however unsure if this included the 

control
7

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

Gibble & Baer 

2011 / E153856

X. laevis / 

Nasco / TP 

(NF49/50)-M 

(90 d total)

T/ 

98 or 

F/ 

40.8

DMSO 

(0.0173) / 

(Y/Y); N 

(Tech), 

NS 

(Form)

NS

1g/L [per 

day –

reviewer 

assume] 

for T / NS 

for F; 

7X/day or 

3X/wk

Metam

Gro

Surv

Imm

--

--

--

986 (N)

--

--

--

>986

Specific parameters within these 

endpoints not reported for 

metamorphosis, growth, survival

Peptide activity (expressed as total 

protein)

Atrazine measured in solvent control (exposed for 

potentially 48 hours) in atrazine technical study; 

survival not reported; study design (replicates, # 

organisms) not reported for the formulation; 

appears frogs used as experimental unit, not tanks, 

for peptide assay for tech atrazine (unknown for 

formulation)

Invalid for growth, survival and metamorphosis 

based on lack of reporting results and control 

contamination; Invalid- for peptide bioactivity 

analyses for atrazine formulation based on lack of 

test design 

Goleman & Carr 

2003 / 45867707

X. laevis / 

Xenopus 

Express 

(Adults) / TP 

(48 hph)-NF66 

(78 d total)

T/ 

98.6

ethanol 

(0.0025)-

E2 group 

only / 

(Y/Y);N

glass

Varied 60-

65 TP 

/0.250 –

4L; 50% 

every 72 

hr

Metam

Gro

Behav

Sexdev

Surv

19.5 (M)

19.5

10.3

10.3

19.5

>19.5

>19.5

19.5

19.5

>19.5

Forelimb emergence, tail resorption

Mass; SVL

↑abnormal swimming

↑ ovotestes

% mortality

Water quality concerns; lack of response to 

positive control and potential delayed 

development 

Invalid based on appeared lack of response to 

positive control and potential overall delayed 

development in the study

Gunderson et al

2011 / E153613

R.catesbeiana / 

Field / TP (NS) 

for 2 d

T/ 

NS

IPA 

(0.001) / 

(N/Y); NS

glass

0.63 g/L; 

static 

(48hr 

exposure)

Bio/Mol

Surv

10

--

>10

--

Relative fold difference for CYP19 

and SF-1

Reported no mortalities in study not 

reported in terms of LOEC/NOEC

Limited water quality reported; while no sign diff 

in CYP19 and SF-1 for each treatment, correlation 

of CYP19 and SF-1 m RNA in the brain when 

combining 1 & 10 ppb resulted in sign diff. 

Invalid based on use on solvent choice (not 

recommended solvent) and no negative control

used in study

Haridon et al. 1993 

/ E4324

Pleurodeles 

waltl / LV for 

12 d

T / 

NS

DMSO 

(0.05) / 

(NS/NS); 

NS

glass

15 LV / 

1.5L; 24 

hr

Bio/Mol

Surv

300

NS

>300

NS

Micronuceated RBCs (genotox)

Reported that maximum 

concentration was dose not to induce 

detectable physiological disturbances 

for 12 days of treatment

Limited water quality reported; Used DMSO at 

higher than recommended conc.; uncertain in what 

the control was comprised of; uncertain if used 

pseudoreplication in results 

Invalid due to uncertain if only one replicate used 

(pseudoreplication

Hayes 2002 / 

E70207

X.laevis / Lab / 

TP(NF46)-M 

(NF66); A for 

46 d

T/ 

NS

EtOH 

(0.004) 

(N/Y);NS

13

plastic 30 TP /4L; 

72 hr

Metam

Gro

Sexdel

Bio/Mol

Surv

200 (N)

200

0.01;0.8

<25

200

>200

>200

0.1;1

25

>200

Time to metamorphosis

Mass; SVL

↑Ovotestes; ↓Laryngeal dilator

↓Plasm testosterone

Mortality

Limited water quality reporting; use of plastic test 

vessels; indirect measured of effect (laryngeal); in 

report results on metamorphosis, growth and 

survival not reported in study

 

13 Study indicates concentrations confirmed by two laboratories; however, unclear if this included the controls.
8

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

Qualitative – Lower level of confidence based on 

higher loading with limited water quality reporting 

and a renewal period of every 3 days

Hayes et al. 2003 

R. pipiens / 

Field / (48hr 

post hatch)-M

T / 

98

EtOH 

(0.0036) / 

(N/Y)

NS14

NS 30 TP / 

4L; 72 hr

Metam

Growth

Surv

Sexdev

NS

NS

NS

<0.1

NS

NS

NS

0.1

Results not presented in report for 

metamorphosis, growth, survival

↑ male gonadal dysgenesis 

(underdeveloped); Sex reversal

No statistical results associated with sexual 

development endpoints ; study reported at 

metamorphosis growth measurements taken but 

results not presented in study

Qualitative – Lower level of confidence based on

higher loading with limited water quality reporting 

and a renewal period of every 3 days 

Hayes et al 2006a

X.laevis / Lab / 

TP (4d post 

hatch)-M 

(NF66)

T/ 

NS

MeOH 

(0.004) / 

(Y/Y); NS

plastic 30 TP/4L; 

72 hr

Sexdev Uncertainty if animals exposed to atrazine in this study or 

if from another study. May have used different clutches 

to make comparisons

Sex ratio of controls skewed; lack of a consistent 

and clear dose response; limited water quality 

parameters reported.

Invalid due to uncertainty if animals were exposed 

to atrazine in this study or were from another study

Hayes et al. 2006b / 

E85815

R..pipiens / 

Field (Adult)/ 

TP (48hr post 

hatch)-G46; X. 

laevis /Lab /A 

for 27 d

T/ 

98

EtOH 

(0.0036) / 

(N/Y); N

plastic 30 TP /4L; 

72 hr

Metam

Gro

Imm/inf

Surv

0.19 (M)

<0.19

<0.19

0.19

>0.19

0.19

0.19

>0.19

Time to metamorphosis 

↓ Mass; SVL

↑ Thymic plaques (X. laevis)

Mortality

Water quality parameters not reported; reported 

results are inconsistent with the data presented in 

report; authors rely on the lack of statistical 

significance to draw conclusions.

Qualitative –Lower level of confidence based on 

higher loading with limited water quality reporting 

and a renewal period of every 3 days as well as 

inconsistent reporting adding to level of 

uncertainty

Hayes et al 2010 / 

E153853

X.laevis / Lab / 

H-A (up to 3 

yrs post M)

T/ 

NS

EtOH 

(0.003) / 

(N/Y); NS

plastic 30 TP/4L; 

72 hr

Sexdev

Bio/Mol

Behav

Surv

Metam

Gro

<2.5(N)

<2.5

<2.5

NS

NS

NS

2.5

2.5

2.5

NS

NS

NS

↓ Breeding gland, laryngeal 

development

↓ Testosterone

↓Mating behavior

Results on survival, metamorphosis 

and growth of animal not reported

In general, the study does not describe and/or 

account for all of the animals used in each 

treatment/replicate; uncertainty in reported results 

(e.g., # of phenotypic female/genotypic male 

compared to total exposed); Results for survival, 

metamorphosis and growth not reported

Qualitative – Lower level of confidence based on 

higher loading with limited water quality reporting 

and a renewal period of every 3 days and 

uncertainty in reported results add to level of 

uncertainty; Invalid for survival, growth and 

metamorphosis

 

14 Study indicates concentrations verified; however, unclear if this included controls
9

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

Hecker et al 2005a

/ E79287

X.laevis / 

Xenopus 

Express / A for 

36 d

T/ 

97.1

NA 

(Y/NA); 

N

NS

1 

frog/10L; 

3 days

Sexdev

Bio/Mol

Bio/Mol

Surv

258(M)

24.6

258

--

>258

258

>258

--

Gondal abnormalities

↓ Plasma testosterone

Aromatase, estradiol, CYP19

4 total mortalities (65 exposed) 

reported; mortality nor presented in 

terms of NOAEC/LOAEC 

Uncertainty in methods for weights (and it 

cascading effect) and gonad evaluation; LOD/LOQ 

not reported for steroid analyses; unclear about 

sample size for steroid analyses

Qualitative-Lower level of confidence due to 

uncertainty in methodology for weights, gonad 

evaluation and biochemical analyses

Hecker et al 2005b

/ E79288

X.laevis / 

Xenopus 

Express / A for 

49 d

T/ 

97.1

NA 

(Y/NA); 

N

fiberglass 4.9 g/L;3 

days

Sexdev

Bio/Mol

Surv

<11.6 M

107

--

11.6

>107

--

GSI

Plasma testosterone; estradiol

1 total mortality reported; mortality 

not presented in terms of 

NOAEC/LOAEC

No solvent control for positive control; Some 

water quality reported but no reporting of nitrates, 

nitrites, and ammonia; limited sensitivity for 

aromatase assay

Qualitative – Lower level of confidence based on 

higher loading with limited water quality reporting 

and a renewal period of every 3 days

Howe et al 1998 / 

MRID 45202910

R. pipiens, B. 

americanus / F / 

TP (G29 & 40) 

for 96 h

F / 

40.8

NA 

(Y/NA); 

N

glass 20 TP / 

15L

Surv 96 hr 

LC50

-- -- LC50 values

R. pipiens (early stage) = 47600

R. pipiens (late stage) = 14500

B. americanus (early) = 26500

B. americanus (late) = 10700

Control survival not reported; no raw data; tested 

above water solubility

Qualitative – Medium level of confidence based on 

lack of control data and only LC50 values reported

and testing above water solubility

Houck & Sessions 

2006 / E117318

R. pipiens / NS 

/ A for 4 or 8 

wks

NS

NS / 

(NS/NS); 

NS

glass NA; 3 

days

Imm/inf

Imm/inf

Surv

<1(N)

10

--

>10

>10

--

↓ plaques representing antibodysecreting cells (4 wks)

Number of Lymphocytes

1 mortality (believed 24 exposed) 

reported; mortality not presented in 

terms of NOAEC/LOAEC

No information provided on culturing conditions 

and limited information on experimental 

parameters; uncertainty in whether used a solvent 

or not as test solution preparation not reported; 

uncertainty in source of frogs; frogs were reported 

to not be feeding in trial 2; analytical 

measurements of treatment groups not reported; 

uncertainty in how animals in Trial 1 were used in 

study

Invalid based on frogs not eating in trial 2 and 

overall lack of culturing and experimental 

parameters and uncertainty in reported results in 

Trial 1

Kerby and Storfer 

2009a / E117314

A. tigrinum / 

Lab / LV 

(4wks) for 14 d

T/ 

NS

MeOH 

(0.02); 

(N/Y); NS

plastic

1 LV 

/0.5L; 

1X/week 

Imm/inf

Surv

200 (N)

200

>200

>200

Infection rate

% Survival basedon atrazine alone, 

reported interactive effect (p=0.03) 

atrazine-virus with atrazine slightly 

increasing survival w/o virus and 

slightly decrease survival w/virus 

Uncertainty in affect of dead larvae on measured 

viral load that was used in analyses; limited water 

quality reported

Qualitative-Lower level of confidence given 

higher than recommended loading rate with 

limited water quality reported and a weekly 
10

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

renewal period; Invalid for infection rate as 

summary data not presented

Kiesecker 2002 / 

E68326

R. sylvatica 

/Field /TP 

(G25) for 4 wk

T / 

NS

ACNT & 

DMSO 

(NS); 

(Y,Y); NS

PE 1 TP/3.5L; 

48 hr

Imm/inf

Surv

<3

NS

3

NS

↓ eosinophils ↑ trematode infections

Mortality not reported; based on 

sample size in Figure 2 in report 

survival appears to be 100%

Uncertain if cercariae used for infection trial were 

exposed to atrazine, therefore there is some 

uncertainty in the environmental relevance of this 

scenario; dilution water only group (no exposure 

to cercariae not used; limited water quality 

reported; uncertainty in statistical analysis

Invalid based on lack of dilution water only 

control, also due to environmental relevance with 

different exposure scenarios between tadpole and 

parasite, unsure of statistical analyses adds to 

uncertainty

Kloas 2009a / 

E112914

X. laevis / 

Xenopus I / TP 

(8 dpf) for 83 d 

T / 

NS

NA / 

(Y/NA); 

Y

15

glass

<1 

g/L/day; 

7X/day

Metam

Gro

Sexdev

Surv

100 (N)

100

100

100

>100

>100

>100

>100

Time to metamorphosis

Mass; SVL>10016

Sex ratio; gonadal development

% Survival

Atrazine contamination of controls; marked 

fluctuations in water quality parameters; 

histological endpoint comparisons not made 

relative to amphibian reference

Quantitative based on fact that although 

deficiencies, study design and reporting were 

robust enough to properly evaluate results

Kloas 2009b / 

153612

X. laevis / Lab / 

TP (NF 48 and 

58) for 4 d

T / 

NS 

NS / 

(NS,NS); 

NS

glass

NS (25TP 

/ 10L

tank); 24 

hr

Bio/Mol

Surv

25

NS

>25

NS

Aromatase, LH, FSH, 5-alpha 

reductase

Mortality not reported

Analysis based on n=8, however only two 

independent tanks tested (removed 4 from each 

tank that contained 25 tadpoles at initiation); 

limited water quality parameters reported during 

test, test solution preparation and what constitutes 

the control was not reported

Qualitative – Low level of confidence based on 

lack test design reporting 

Koprivnikar et al

2007 / E103348

R. sylvatica / 

Field / TP 

(G28/29) for ca. 

31 d

F / 

40.8

NA / 

(N17/NA); 

NS

plastic

1 TP 

/1.8L; 72 

hrs

Imm/inf

Surv

3 (N)

30

30

>30

↑ intensity of infection (frogs 

exposed but cercariae not)

Mortality

Control survival was 61.9%; Dilution water only 

treatment (no cercariae) was not used; replicate 

variability not reported; effects only observed 

when tadpoles, not cercariae were exposed to 

atrazine –uncertainty in environmental relevance; 

 

15 Atrazine detected in one out of 4 blocks of negative control tanks in one of the studies; it was excluded from analysis

16 Significant decrease in female weight and SVL observed at 0.01, 1 and 25 for females only but not at 0.1 and 100 ppb in one of the two reported studies; No 

effect on wt and SVL reported in other study; EFED does not consider the differences in wt and SVL to be a concentration-dependent effect

17 A dilution water only control was not included. A control that contained no atrazine but cercariae that were also not exposed to atrazine was used in the study
11

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

limited water quality reported

Invalid based on lack of dilution water only 

control, lack of reported replicate variability and 

adverse effects observed only when tadpoles, not 

cercariae, were exposed (unknown environmental 

relevance) adds to the level of uncertainty

Koprivnikar et al

2010 / E153870

R. pipiens / E 

from CBSC18 / 

TP (<G27)- ~ 

G4319

T/ 

99.2

EtOH 

(100ug/L) 

/ (Y/Y); 

NS

plastic 1 TP/L; 

weekly

Metam

Gro

Surv

<3 (N)

<3

<3

3

3

3

Developmental stage

Mass

Mortality

Variability in mortality across 3 ppb treatment 

groups; uncertain if developmental stage was 

accounted for in mass endpoint; uncertainty in 

reported results in figures; uncertainty if 

organisms were field collected or laboratory reared

Invalid based on uncertainty in reported results 

based on Figures in report and limited reported 

water quality with relatively long renewal period 

and variability in mortality across treatment 

groups add to uncertainty

La Fiandra et al. 

2008 / E103336

H. versicolor / 

Lab / TP (G25) 

-30 d past M

F/ 

88.2

NA; 

(Y/NA); 

NS

glass

3 TP/7L; 

50% 

1X/wk

Metam

Gro

Sexdev

Surv

195 (M)

195

195

195

>195

>195

>195

>195

Larval period

Mass; SUL20

Sex ratio; gonad development

% Survival

Limited water quality reported; uncertainty if 

adults used to obtain eggs for culturing were from 

the field or laboratory

Qualitative-Lower level of confidence, while 

loading was within recommended limit, limited 

reported water quality with relatively long renewal 

period(50% renewal every week) may have 

potential environmental condition issues

Langerveld 2009 / 

E121497

X. laevis / Lab / 

TP (NF43) to 

NF46

T/ 

99.4

NA / 

(Y/NA); 

NS

Pyrex

4 TP/1L; 

every 2-3 

days

Gro

Bio/Mol

Surv

<400 (N)

<400

<400

400

400

400

↓ Mass; SVL

Expression of multiple genes

% Survival

Limited water quality reported; Uncertainty 

regarding overall mortality in control and 400 ppb 

however, substantial mortality observed at 400 

ppb when approaching stage 63, therefore may 

not be unexpected to observe significant sublethal 

changes

Invalid for growth and bio/mol due to significant 

mortality compared to control in the only atrazine 

concentration tested also high loading rate with 

limited water quality and renewal rate periodically 

 

18 CBSC = Carolina Biological Supply Company (Burlington, NC)

19 G43- first observance of reabsorbed tail

20 SUL = snout-urostyle length
12

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

>48 hours; Qualitative Low level of confidence for 

survival based on limited water quality, loading 

and renewal rate.

Larson et al. 1998 / 

E60632

A. tigrinum / 

Field / LV 

(SVL= 30mm) 

for 86 d

T / 

NS

ACTN 

(NS)/ 

(N/Y); Y

PE

1 TP/4L; 

50% at 

48hr

Metam

Gro

Bio/Mol

Surv

<81.8(M

81.8

<81.8

--

81.8

>81.8

81.8

--

↓metamorphosis

Mass; SVL

↑plasma thyroxine;↓ corticosterone

Reported no exposure related 

mortalities; mortality not reported in 

terms of NOAEC/LOAEC

Atrazine measured in controls; lower dissolved 

oxygen reported

Invalid due to control contamination and solvent 

concentration not reported and no negative control 

tested add to uncertainty

Lenkowski & 

McLaughlin 2010 / 

E153851

X. laevis / Lab / 

TP (NF41) for 

6-48 hr

T/ 

NS

DMSO 

(0.1-0.35) 

/ (Y/Y); 

NS

NS

13-18 TP 

/0.01L; 24 

hr (48 hr 

exposure)

Bio/Mol

Surv

350(N)

NS

3500

NS

↑ MMP18 expression Limited reporting on test elements (replicates, 

solution preparation, test vessels, analysis of test 

solutions, water quality parameters, loading, 

control survival)

Invalid due to solvent concentration and solvent 

choice and lack of reporting on test design and 

experimental parameters contributing to higher

level of uncertainty

Lenkowski 2010 / 

E153876

X. laevis / Lab / 

TP (NF41) for 

48 hr

T/ 

NS

DMSO 

(0.1-0.35) 

/ (Y/Y); 

NS

NS

13-18 TP 

/0.010L; 

24 hr (48-

hr 

exposure)

Metam

Surv

350 (N)

--

3500

--

↑ Malformations in intestines and 

cranial cartilage

Reported no mortality in atrazine 

exposures, control mortality not 

explicitly reported

Limited reporting on test elements (solution 

preparation, test vessels, analysis of test solutions, 

water quality parameters)

Invalid due to solvent concentration and choice 

and lack of reporting on test design and 

experimental parameters contributing to higher

level of uncertainty

Lenkowski et al

2008 / E103268

X. laevis / Lab 

/TP (NF41-46) 

for 48 hr

T / 

98

DMSO 

(0.1-0.35) 

/ (Y/Y); 

NS

NS

15 TP 

/0.01L; 24 

hr (48 hr 

exposure)

Metam

Surv

<10,000 

(N)

--

10000

--

↑ visceral bleeding & organ 

malformations

Mortality reported as <5%

Limited reporting on test elements (test vessels, 

analysis of test solutions, water quality 

parameters)

Invalid due to solvent concentration and lack of 

reporting on test design and experimental 

parameters contributing to higher level of 

uncertainty

Oka 2008 / 

E103345

X. laevis / Lab / 

TP (NF49)-M 

(56 d total) 

T/ 

97.4

EtOH 

(0.01) / 

(N/Y); NS

glass

30 TP

/10L; 

3X/wk

Metam

Bio/Mol

Sexdev

Surv

100 (N)

100

1

--

>100

>100

10

--

Time to metamorphosis

Aromatase

↑ female ratio

Reported ≥ survival in controls and 

some treatment groups with low 

survival in one of two tanks for two 

concentrations

Estrogenic activity measured in parental food; 

variable replicate mortality which resulted in only 

one replicate for analysis for two treatment groups 

for sex ratio endpoint; no statistical results 

reported for metamorphosis; limited water quality 

reported

Qualitative- Lower level of confidence based on 

higher loading with limited water quality reporting 
13

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

and an occasional renewal period of every 3 days 

coupled with variable mortality in tanks for two 

treatment groups (not dose responsive); Invalid for 

metamorphosis as statistical results not reported.

Olivier & Moon 

2010 / E153629

A.maculatum / 

Field / E for ~ 

76 d

F/ 

42.6

NA / 

(Y/NA); 

NS

plastic

1 egg 

mass 

/0.750L; 3 

wks

Metam

Surv

50 (N)

50

100

100

Developmental stage before death or 

hatch

% Hatch; mortality reported as egg 

mass mortality specific stats not 

reported

Temperature regulator malfunction in which 

pathologies were observed in treatment groups 

(not control) shortly thereafter and mortality in 

atrazine treatments occurred shortly after solution 

renewal; Uncertainty in number of embryos per 

treatment and evaluation of oxygen depletion; 

limited water quality 

Qualitative-Lower level of confidence as it 

appears that additional potential stressors of 

temperature change and solution renewal may 

have contributed to observed pathologies as well 

as limited water quality reported with long 

renewal period (21 d)

Orton et al 2006 / 

E82034

R.pipiens / 

Carolina 

Biological / 

Taylor-Kollros 

Stage 2/3 –

23.24 

T/ 

98.2

NA / 

(Y/NA); 

N

NS

20 TP /8L; 

75% at 48 

hr

Metam

Gro

Sexdev

Surv

15 (M)

15

<15

<15

>15

>15

15

15

Time for forlimb emergence

SVL

Sex ratio; gonad develop

% mortality; lower in treatment 

groups

High control mortality (ca. 50%); higher incidence 

of intersex (12.5%) in controls; sex ratio of 

controls appears skewed; summary data for 

metamorphosis and growth not presented

Invalid based on higher incidence of reported 

gonadal intersex in control and higher control 

mortality also no summary data for 

metamorphosis and growth

Morgan 1996 / 

E63246

X. laevis / 

Xenopus I

(adults) / E for 

96 hr

F / 

40.8

NA / 

(Y/NA); 

NS21

plastic

NS (20 

E/petri 

dish) ; 24 

hr

Metam

Surv 

-- 11000 

or 1100

LC50 = 100000 or 126000 

EC50 (abnormalities) = 33000 or 

<8000 

Limited water quality reported, test solution fill 

volume not reported; conducted above water 

solubility; formulation used so source of toxicity 

unknown

Qualitative – Medium level of confidence based 

on limited water quality reported 

Rohr et al., 2003/ 

E71723

A. barbouri / 

Field / E for 37 

d (53 d total 

test)

T / 

80

DMSO 

(0.03) / 

(Y/Y); NS

Glass

10 LV/2L; 

50% at 48 

hr

Gro

Behav

Surv

400 (N)

40

400

>400

400

>400

Mass; SVL; limb deformaties

↑Locomotor activity

Total Survival 

Solvent control contained both acetone and 

DMSO whereas atrazine concentrations only 

contained DMSO (which is not a recommended 

solvent); author states organisms likely exposed to 

atrazine prior to test due to agricultural activities 

around collection site; limited water quality 

 

21 Test concentrations measured but do not report results for control; unsure if analyzed
14

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

reported; uncertain if potentially different stages at 

test termination (stages not reported) influenced 

growth analysis; mass summary data not presented

Qualtiative- Lower level of confidence based on 

higher than recommended solvent concentration 

using a non-recommended solvent in addition the 

limited reported water quality with the higher

loading rate add to the level of uncertainty; Invalid 

for mass as summary data not presented

Rohr et al., 2004 / 

E81748

A. barbouri / 

Field / E-M 

(mean 64 d)

T / 

80

DMSO 

(0.03) & 

ACTN 

(0.03)22 / 

(N/Y); N

NS

31 or 40 

E/9.5L; 

weekly

Metam

Gro

Behav

Surv

4(N)

40

40

40

40

400

400

400

↑ time to metamorph

↓ mass

↑ locomotion activity; ↓refuge use

↓ Survival

White film on dead embryo unknown if fungus 

caused or followed mortality; larval survival 

differences between years; although pH, temp, and 

DO ok, nitrates and ammonia not reported; 

Qualitative-Lower level of confidence based on 

higher than recommended solvent concentration 

using a non-recommended solvent in addition to 

uncertainty in the source of mortality in the dead 

embryos

Rohr and Palmer, 

2005 / E81664

A. barbouri / 

Field / 

Metamorphs 

for 5 hrs

T / 

80

DMSO 

(NS) / 

(N/Y); NS

NS NA; 5 hr 

exposure

Behav

Surv

4(N)

--

40

--

↓ water conserving behavior; ↑ 

activity

Reported no mortality; results not 

presented in NOAEC/LOAEC terms

This study used metamorphs from previous study 

(Rohr et al 2004, E81748) in which organisms 

were exposed to different atrazine concentrations 

and test subjects in this study were not exposed to 

any additional atrazine during the behavioral study 

(water conserving behaviors). See Rohr et al 2004 

(E81748) for comments

Qualitative-Lower level of confidence based on 

identified uncertainties in the other study from 

which the organisms in this study were obtained 

Rohr 2008b / 

E112912

R. clamitans / 

Field / TP 

(G24-26) for 14 

d

T/ 

>98

ACTN

(NS) / 

(Y/Y); NS

glass 4 TP 

/0.3L; NS

Imm/inf

Surv

<201 

(M)

201

201

>201

↑ % of encyted cercariae

Mortality

Tadpoles exposed to atrazine before and after 

cercariae exposure but cercariae not-uncertainty in 

environmental relevance; for cercariae-tadpole 

results, the treatments are compared to the solvent 

control suggesting a significant difference between 

the controls; limited water quality reported; 

control mortality not clearly indicated

Qualtiative- Lower level of confidence as control 

 

22 Solvent concentration was calculated by reviewer
15

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

mortality not clearly reported limited water quality 

reported with a non-specified renewal period 

(tadpoles exposed for 7 days at a time) and higher

than recommended loading and environmental 

relevancy that adds to the uncertainty

Rohr et al. 2009 / 

E117315 

B. americanus / 

Field / LV 

(G25-27) for 4 

d

T/ 

99

ACTN 

(0.0002) / 

(N/Y); NS

NS

6 TP/0.5L 

(96hr) 

then 

1/3.5L; 

static

Behav

Surv

196 (M)

--

>196

--

Fear queues (predator and parasite); 

activity

Reported no effect on mortality with 

nearly 100% survival

No negative control (authors report no effect from 

solvent based on previous data); limited water 

quality reported

Qualitative-Higher level of confidence, however 

no negative control and limited water quality

Rohr et al., 2006 / 

E82038

A. barbouri / 

Field / A

T / 

80

DMSO 

(0.03) & 

ACTN 

(0.03)23 / 

(N/Y); N

NS

31 or 40 

E/9.5L; 

NA 

Surv <4 4 ↓ Long term survival (carryover 

effects)

This study used metamorphs from previous study 

(Rohr et al 2004, E81748) in which organisms 

were exposed to different atrazine concentrations 

and test subjects in this study were not exposed to 

any additional atrazine to observe survival due to 

carryover effects. See Rohr et al 2004 (E81748) 

for comments

Qualitative-Lower level of confidence based on 

identified uncertainties in the other study from 

which the organisms in this study were obtained 

Rohr et al. 2011 / 

E153803

A.barbouri / 

Field / E-H 

(49d) or LV 

(NS)-M or 19 d

T/ 

99

ACTN 

(8.25) / 

(N/Y); NS

glass 12 E/2L; 

weekly

Gro(E)

Metam 

(L)

Gro (L)

Surv

40 (N)

NS

NS

<4

400

NS

NS

4

Mass

Authors indicate effect on metam 

and growth but conc. not reported

Embryos – ↑ Mortality; larval 

experiment- no significant for 

atrazine treatment group

Larval survival lower across all treatments and 

control for study at 19°C; larval mass from 

embryo study conducted using preserved 

organisms; limited water quality reported

Invalid based on higher than recommended 

solvent concentration using a non-recommended 

solvent in addition to not using a negative control 

group; also limited water quality reporting with 

higher than recommended loading and weekly 

renewal period add to the level of uncertainty

Spolyarich et al. 

2010 / E153638

L.tasmaniensis 

/ Field / TP 

(G28) to G42 

(4wks)

T/ 

98

NA / 

(Y/NA); 

N

glass

10 

TP/10L; 

2X/wk

Metam

Gro

Sexdev

Surv

30 (N)

30

30

--

>30

>30

>30

--

Rate of development

Length

Sex ratio; gonad development

Reported 2.3% mortality in test 

overall; mortality not reported in 

terms of NOAEC/LOAEC

Sex determination based on gross morphology; 

histology on male gonads only; Metamorphosis 

and length data analysis appear to be based using 

the individual frog as the experimental unit and 

not the tank as data in figures appears to be on 

pooled results of the three trials

 

23 Solvent concentration was calculated by reviewer
16

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

Qualitative-Higher level of confidence as loading 

seemed within recommended levels, however, 

ammonia and nitrate concentrations not reported 

with a 2X/wk renewal period and field collected 

organisms (Sydney Australia suburb) so prior 

exposure history is unknown

Storrs and 

Kiesecker 2004 / 

E78290

Pseudacris 

crucifer, R. 

sylvatica, B. 

americanus, R. 

clamitans / 

Field / TP 

(early stage 

G25-27) & (late 

stage G29-36) 

for ca. 30 d

F / 

85

NA / 

(Y/NA); 

NS

PE

1 TP/ 0.1 

or 0.5L; 

every 3 

days

Surv NDR 2.84 Survival- not dose responsive, for all 

but wood frog, report sign. effects at 

2.84, but not always at 25 or 64 

Survival not dose- responsive; animals that 

metamorphosed during the experiment were 

eliminated from the data set; limited water quality 

reported

Qualitative-Lower level of confidence due to 

survival response not dose-responsive and limited 

water quality reported with loading and renewal 

rate.

Storrs & Semlitsch 

2008 / E103378

H. versicolor; 

R. 

sphenocphala; 

B. americanus / 

Field / TP 

(G25)-

Metamorphs 

T/ 

99.2

EtOH 

(0.0125) / 

(Y/Y); N

Glass & 

plastic

1 TP/ 

0.8L; 72 

hr

Metam & 

Sexdev 

(H. vers.)

Metam & 

Sexdev

(R. sphen)

Metam & 

Sexdev

(B. amer)

Surv (all)

25.1 (M)

30.4

125

NS

>25.1

>30.4

>125

NS

Time to metamorphosis; Ovarian 

stage

Time to metamorphosis; Ovarian 

stage

Time to metamorphosis; Ovarian 

stage

Mortality not reported

Limited water quality reported; very slight 

estrogenic activity measured in dilution water; 

Uncertainty in number of animals exposed

Qualitative-Lower level of confidence based on 

limited water quality reported with slightly higher

than recommended loading and renewal period of 

72hr as well as slightly higher than recommended 

solvent concentration

Storrs Mendez et 

al. 2009/ E118898

B. americanus / 

Field / 

Metamorphs for 

2 d 

T/ 

99.2

ACTN 

(NS)/ 

(NS/NS); 

NS

NS NA; NA

Behav

Surv

1430

μg/kg(N)

NS

>1430

NS

Soil choice preference (ATZ or no 

ATZ)

Limited reporting on control group preparationuncertainty regarding whether control contained 

solvent; uncertainty in measured conc. due to less 

than nominal and long storage time

Qualitative-Medium level of confidence based on 

limited reporting for control preparation 

Storrs-Mendez & 

Semlitsch 2009/ 

E153841

H. versicolor; 

R. 

sphenocphala; 

B. americanus / 

Field / TP 

(G25)-

Metamorphs 

and TP (G25)-J 

(4 or 6 mnths)

T/ 

99.2

EtOH 

(NS) / 

(N/Y); N

NS

1 TP/0.8L 

or 

20TP/20L; 

72 hr

Sexdev 

(L)

B. amer

R. sphen

H. versi.

Sexdev (J)

B. amer.

R. sphen

H. versi 

Surv All

7.55(M)

0.8

<0.92

<3

29.5

24.6

NS

124

3

0.92

3

>29.5

>24.6

NS

Sex ratio for all endpoints

Mortality not specified Results on larvae appear to be same as reported in 

Storrs & Semlitsch 2008 / E103378; Results not 

dose responsive between treatments and time; 

Uncertainties about test design for juveniles

Qualitative-Lower level of confidence; see 

comments for Storrs & Semlitsch 2008 / E103378
17

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

Sullivan & Spence 

2003

X.laevis / 

Xenopus 

I(Adults) / 

TP(NF46-48)-

M

T 

/99

ACTN 

(0.0032) / 

(Y/N); NS

glass

6 TP/0.5L 

starting 

varied 

over 

time;every 

3-4 days 

Metam

Gro

Surv

40

<20

320

320

20

>320

↑ Time to metamorphosis w/nitrate 

↓Mass at metamorphosis; no effect 

on metamorphosis for this study

Reported mean mortality for entire 

atrazine only study (p value not 

reported); when atrazine w/nitrite, 

mortality sign. greater for nitrate 

with 320 μg/L atrazine than 0 μg/L 

nitrate

Changing exposure conditions and alterations in 

the number of replicates; 

Qualitative-Lower level of confidence based on 

changing exposure conditions and alterations in 

number of replicates

Tavera-Mendoza et 

al 2002 / E60639

X.laevis/Xenopu

s I/ TP(NG56) 

for 48 h 

T/ 

99

NA / 

(Y/NA); 

N

glass

16 TP/ 

15L; static 

(48hr 

exposure)

Sexdev

Surv

<18

--

18

--

↓ testes size, spermatogonial nests 

and nurse cells 

Reported no mortalities in control or 

treatment; mortality not reported in 

terms of NOAEC/LOAEC

Need documentation of the measurement 

endpoints, elaboration on the ecological relevancy 

of the measurement endpoints, and clarification of 

the number of exposed animals; appeared to use 

individual animals not replicates as experimental 

unit

Invalid based on uncertainty if results based on 

pseudoreplication

Tavera-Mendoza et 

al 2002 / E77830

X. laevis 

/Xenopus I / 

TP(NF56) for 

48 h

T / 

99

NA / 

(Y/NA); 

N

glass

16 

TP/15L; 

static (48 

hr 

exposure)

Sexdev

Surv

<18

NS

18

NS

↓ primary oogonia ↑ secondary 

oogonia ↑ atresia 

Survival not specified but based on 

sample size it appears to be 100%

Only one dose; uncertainty of the nature of the 

measurement endpoints, and the relevancy of the 

endpoint to higher order measures of organismal 

fitness; used individual animals not replicates as 

experimental unit

Invalid based on uncertainty if results based on 

pseudoreplication

Villeneuve et al. 

2003 / 45867708

X. laevis / / A 

for 26, 43 or 47 

d

T / 

NS

NA / 

(Y/NA); 

NS

fiberglass

15 A 

/120L; 

50% every 

3 days

Bio/Mol

Surv

25 (N)

--

>25

--

Gonad and brain aromatase

Mortality ranged from 3-19% and 

was not presented in terms of 

NOAEC/LOAEC

Lack of replication in some studies; observed 

variability in endpoints; potential water quality 

issues

Invalid for studies (male) due to no replication; 

Qualitative-Lower level of confidence in 

remaining study (female) due to higher variability 

in endpoint in control

Wan et al. 2006 / 

E89626

R. catesbeiana 

/Ward’s Natural 

Science / TP 

(10cm) for 96

hr

T / 

98 & 

F / 

48.5

ACNT 

(NS); 

(Y/Y); NS 

(dilution 

water 

analysed)

glass 0.8 g/L; 

static

Surv -- -- Technical

96 hr LC50 > 16000

Formulation

96 hr LC50 > 480000 (232800 ai)

Solvent concentration not reported; raw data not 

provided, control mortality not reported

Qualitative – Medium level of confidence based 

on lack of summary data and no reported solvent 

concentration

Williams & 

Semlitsch 2010 / 

E153825

B.americanus 

& P. triseriata 

& H. versicolor

F / 

48.8

NA / 

(Y/N); N

Glass 

&plastic

NS (1 

TP/1L 

container); 

Metam

Gro

(all 

1.25(M)

1.25

>1.25

>1.25

Time to metamorphosis

Mass

Limited water quality reported; fill volume of test 

vessel not reported (1 TP /1L container); mass 

summary data not presented
18

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

/ Field / TP 

(G25)-M

72 hr species)

Surv 1.25 1.25 % Surviving

Qualitative- Lower level of confidence based on 

limited water quality reported with a renewal 

period of 72 hrs and probably slightly higher than 

recommended loading; Invalid for mass as 

summary data not presented

Zaya et al. 2011a /

E153847

X.laevis / NS 

(Nasco?) / TP 

(NF47)-NF 

58or 62

T/ 

NS

NA / 

(Y/NA); 

NS

glass 4 TP/1L; 

3X/wk

Metam

Gro

Behav

Sexdev

Imm/inf

Surv

200 (N)

25

400

400

200

400

400

200

>400

>400

400

>400

Time to metamorphosis

Mass; fat bodies

Feeding

Sex ratio (gross morphology)

↑ Activated caspase 3 immuno(+) 

cells

Survival

Potential effects of stress during weighing and 

staging during study; uncertainty about protocol 

for feeding endpoint; variability in fat body and 

liver size endpoints; limited water quality reported

Qualitative-Lower level of confidence based on 

limited water quality reported with a higher than 

recommended loading and a renewal period of 72 

hours

Zaya et al 2011b / 

E153848

X.laevis / NS 

(Nasco?) / TP 

(NF47)-NF 

58or 62

T/ 

NS

NA / 

(Y/NA); 

NS

glass 4 TP/1L; 

3X/wk

Bio/Mol

Surv

<400 (N)

400

400

>400

Changes in gene expression (actylCoA ehydrogenase and 

glucocorticoid receptor)

Used organisms from Zaya et al

2011a (E153847)

etiology of the effects on gene expression reported 

in this article are uncertain; potential bias in 

sampling; limited water quality reported 

Qualitative-Lower level of confidence based on 

potential biasing and limited water quality 

reported with a higher than recommended loading 

and a renewal period of 72 hours

Cosms

Boone & BridgesBritton 2006 / 

E88290

Hyla. versicolor

/ Field / T 

(G25)- M (35 

d)

F / 

40.8

NA / 

(Y/NA); 

NS

PE 30 TP 

/1000L

Metam

Gro

Surv

20 (N)

20

20

>20

>20

>20

Days to metamorphosis

Mass

Survival

Low power to detect differences; 

Qualitative

Boone & James 

2003 / E81455

R.

Sphenocephala

B. americanus

A. maculatum 

& A. texanum / 

Field / LV for 

39-41 or 80 d

F/ 

40.8

NA / 

(Y/NA); 

NS

PE 20 or 60 

TP/1000L

Metam

Gro

Surv

197 & 

<197

<197

197

>197 & 

197

197

>197

↑ time to metamorphosis for A. 

texanum; no effect other species

↓Mass (all species)

Survival

Limited data to interpret atrazine only effects for 

some endpoints/scenarios; lack of analytical 

measurements in dilution water

Qualitative

DeNoyelles 1989 / 

47543608

R. catesbiana / 

NS/ Stage NS 

for 805 d

F / 

41 & 

T/ 

97

NA / 

(Y/NA); 

NS

NS NS

Gro

Surv

<20 (N) 20 ↓ overall biomass Bullfrogs immigrated to ponds-uneven distribution 

likely; potential chemicals in ponds prior to testing 

unknown

Qualitative

Detenbeck et al. R. pipiens / F/ 85 NA / Wooden 20 TP Metam 25 (N) >25 % adult appearance; hind limb length Control contamination; short acclimation periods; 
19

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

1996 / E16839 Field / TP (NS) 

for 41 d

(Y/NA); 

Y

cage 

w/plastic

/cage/ 

flowering 

stream

Surv NS NS Not specified higher variability (lower power) 

Qualitative based on review for plants

Diana et al 2000 / 

E59818

H. 

versicolor/ 

Field / TP to 

metamorphs

T/ 

NS

ACTN 

(0.04% )/ 

(N/Y); Y

plastic 40 LV 

/90L

Metam

Gro

Surv

2000

20

2000

>2000

200

>2000

Time to metamorphosis

↓Mass; SVL

% Survival

Control contamination; pre-metamorphosis weight 

and length were not determined

Qualitative based on review for plants

Du Preez et al 2008 

(lab and cosm) / 

E103271

X laevis / Field 

/ TPto A (2 yrs 

post metam)

T / 

NS

MeOH 

(0.025) / 

(N/Y); N

NS

75 

frogs/2,50

0L

Metam

Sexdev

Surv

24.8

24.8

24.8

>24.8

>24.8

>24.8

Time to metamorphosis

Gonadal development; sex ratio

% Survival

Continuation of Jooste 2005 (46965603); Temp 

concerns; uncertainty about adding tadpoles at 

different times effect on metamorphosis endpoint; 

higher occurance in testicular oocytes in controls

Invalid based on control oocytes

Jooste et al 2005 / 

MRID 46965603

X.laevis / Field 

(Adults) / TP -

M

T/

MeOH 

(0.025%) / 

(N/Y); 

NS24

PE 800 TP / 

1100L 

Metam

Sexdev

Surv

30.8 (M)

30.8

NS

>30.8

>30.8

NS

Time to metamorphosis

Testicular oocytes

Preliminary to DuPreez 2008 (E103271); 

Unusually long development times; feeding and 

temperatures may have limited development. 

Higher incidence of testicular oocytes in controls

Invalid based on control oocytes

Langlois 2010 / 

E120046

R.pipiens / 

Field / 

TP(G27)-

M(G42)

F / 

(NS)

NA / 

(Y/NA); 

Y

HDPE 0.1 g/L

Metam

Sexdev

Bio/Mol

Surv

<0.1

0.1

0.1

0.1

0.1

1.8

1.8

1.8

↓ Metamorphic success

↑ Sex ratio (female)

↑ and ↓Brain eralph and tail doi3

Total survival

Control contamination and atrazine detected in 

area where organisms collected; uncertainty in 

biochemical responses on gross endpoints; 

metamorphic success and sex ratio based on total 

exposed, no replicate variability reported

Qualitative

Relyea 2009 / 

E114296

H.versicolor &

R. pipiens / NS 

(collected as 

eggs) / TP-M

T/ 

99

EtOH 

(0.003) / 

(Y/Y); 

NS25

cattle 

watering 

tanks

9 TP/m2

H. vers

Metam

Gro

Surv

R. pipiens

Metam

Gro

Surv

6.4

<6.4 

6.4

6.4

6.4 

6.4

>6.4

6.4

>6.4

>6.4

>6.4

>6.4

Time to metamorphosis

↑Mass

Survival

Time to metamorphosis

Mass

Survival

Atrazine concentrations were not monitored 

throughout the experiment, although an initial 

measured value was reported. Control tanks were 

not analyzed for atrazine and the other nine 

pesticides tested. Additionally, it is unclear how 

much ethanol was added to atrazine-only treatment 

tanks.

Qualitative 

Rohr and Crumine 

2005 / E117319

R.sylvatica/ 

Field / TP-M

F / ACTN 

(0.00246) PE 20 TP /10L

Metam

Gro

50

50

>50

>50

Stage at test termination

Mass

Atrazine concentrations not monitored; unclear 

acclimation periods; prior exposure for tadpoles

 

24 Atrazine measured in treatment tanks; unclear if analyzed in control

25 Controls not analyzed; however, dilution water prior to addition to tanks was analyzed and found to be free of atrazine
20

Citation / 

ECOTOX(E)/ 

MRID

Species / 

Source / 

Exposure 

Period (stages 

& test 

duration)1

TS2/ 

% ai

Solvent 

(%)3/ 

NC/SC 

(Y/N); 

MA in 

CNT 

(Y/N/NS)4

Test 

vessel 

material5

Loading; 

Renewal 

Period6

Reported Results7

Indentified Uncertainties / Concerns and 

Classification

Endpoint NOEC

(μg/L)

LOEC 

(μg/L)

Effect

/ (Y/Y); 

NS26

Behav

Surv

<50

--

50

--

↓% of hiding

Reported no dead tadpoles found 

during study; mortality not presented 

in terms of NOAEC/LOAEC

Qualitative

Rohr et al. 2008a / 

E112910

R.pipiens & R. 

clamitans / 

Field/ TP for 4 

wks

T/ 

98

NS (Y/Y); 

NS27 plastic

65 

amphibs/8

00L

Imm/Inf

Surv

<117

117 

&<117

117

117 

&>117

↓ eosinophils ↑ trematode cycts (R. 

clam);

↓Melanomacrophage

↓ % survival for R. pipiens and no 

effect for R. clamitans

Unknown solvent and concentration. Atrazine 

concentrations were not monitored during test. 

Unknown if in algal communities similar at test 

initiation . 

Qualitative

 

26 Atrazine measured in treatment tanks; unclear if analyzed in control

27 Atrazine measured in treatment tanks; unclear if analyzed in control